The C2H2 Transcription Factor Con7 Regulates Vegetative Growth, Cell Wall Integrity, Oxidative Stress, Asexual Sporulation, Appressorium and Hyphopodium Formation, and Pathogenicity in Colletotrichum graminicola and Colletotrichum siamense

The Colletotrichum genus is listed as one of the top 10 important plant pathogens, causing significant economic losses worldwide. The C2H2 zinc finger protein serves as a crucial transcription factor regulating growth and development in fungi. In this study, we identified two C2H2 transcription factors, CgrCon7 and CsCon7, in Colletotrichum graminicola and Colletotrichum siamense, as the orthologs of Con7p in Magnaporthe oryzae. Both CgrCon7 and CsCon7 have a typical C2H2 zinc finger domain and exhibit visible nuclear localization. Disrupting Cgrcon7 or Cscon7 led to a decreased growth rate, changes in cell wall integrity, and low tolerance to H2O2. Moreover, the deletion of Cgrcon7 or Cscon7 dramatically decreased conidial production, and their knockout mutants also lost the ability to produce appressoria and hyphopodia. Pathogenicity assays displayed that deleting Cgrcon7 or Cscon7 resulted in a complete loss of virulence. Transcriptome analysis showed that CgrCon7 and CsCon7 were involved in regulating many genes related to ROS detoxification, chitin synthesis, and cell wall degradation, etc. In conclusion, CgrCon7 and CsCon7 act as master transcription factors coordinating vegetative growth, oxidative stress response, cell wall integrity, asexual sporulation, appressorium formation, and pathogenicity in C. graminicola and C. siamense.


Introduction
The Colletotrichum genus, comprising 200 members, is listed as one of the top 10 important plant pathogens, and can infect more than 3000 species of monocotyledons and dicotyledons, causing great economic losses all over the world [1,2].Colletotrichum graminicola is the pathogen causing anthracnose leaf blight and stalk rot in maize, which has led to serious damage to the maize industry in recent years [3].C. graminicola usually produces two kinds of conidia, namely, falcate conidia and oval conidia [4].Oval conidia are generally formed inside the host and primarily related to intra-tissue spread [4].Falcate conidia are primary infective spores that can individually produce germ tubes and then form appressoria to penetrate the host [5].C. siamense, which is a member of the C. gloeosporioides species complex, is the main causal agent of rubber anthracnose [6].C. siamense invades the epidermis of rubber tree leaves, posing a threat to the growth of rubber trees and natural rubber production [6].Like the falcate conidium of C. graminicola, the conidium of C. siamense generates a germ tube and forms an appressorium at its top after adhering to leaves.Then, the appressorium produces a penetration peg and invades the epidermis of leaves [7].In some Colletotrichum species, the hyphopodium is usually developed at mature hyphal tips, which also plays a role in the penetration of the plant epidermis [8].Therefore, conidia serve as a primary dissemination tool and play an important role in the infection of the two pathogenic fungi [9,10].In-depth analyses of the regulatory mechanisms of conidial production and infection have important theoretical and practical significance for understanding the interactions between pathogens and hosts and thus potentially for effectively controlling diseases.
For many pathogenic fungi, the colonization in hosts usually comprises conidial adhesion, germination, appressorium formation, and invasive growth, which is precisely regulated by transcription factors (TFs) [11].The C 2 H 2 zinc finger protein is a common TF, and plays a vital role in coordinating growth and development in fungi [12].Con7-like TFs are typical C 2 H 2 zinc finger proteins.Con7 was first characterized in Magnaporthe oryzae, where con7 mutants produced abnormal conidia with significantly reduced conidiation [13].Further research showed that Con7p was also required for appressorium formation in M. oryzae [14,15].In Verticillium species, the Con7 ortholog Vta2 is a positive regulator of vegetative growth, conidiation, H 2 O 2 detoxification, and virulence [16].In Fusarium oxysporum, deleting con7-1 resulted in non-pathogenic mutants, which also exhibited defects in hyphal branching, asexual sporulation, and cell wall structure [17].In F. graminearum, disrupting FgCON7 also led to severe defects in growth, asexual/sexual development, and virulence [18].Overall, the Con7-like TFs act as central regulators in the processes of growth, development, stress response, and infection.
In this study, two Con7-ortholog TFs, CgrCon7 in C. graminicola and CsCon7 in C. siamense, were functionally characterized through genetic manipulations.We found that both CgrCon7 and CsCon7 were implicated in regulating vegetative growth, oxidative stress response, cell wall integrity, conidiation, appressorium formation, and virulence.Furthermore, the regulatory networks of the two TFs were also elucidated by transcriptome analysis.

Fungal Strains and Culture Conditions
In this study, C. graminicola strain CgM2 (CgrWT) and C. siamense strain CsWT were used as wild-type strains and preserved in the School of Life and Health Sciences, Hainan University.The wild-type strains and their derivative strains were routinely maintained on potato dextrose agar (PDA) for 5 days at 28 • C and then stored at 4 • C. The media used in this work, such as PDA, CM, MM, CZAPEK (CZ), LB, DCM, and TB3, were prepared as described previously [19].To evaluate vegetative growth, all strains were inoculated on PDA, CM, CZ, and MM media at 28 • C, and colony diameters were measured 5 days post-incubation (dpi).

Nucleic Acid Manipulations and Sequence Analysis
The genomic DNA (gDNA) of C. graminicola and C. siamense was obtained using the CTAB method [20].Total RNA extraction and cDNA synthesis were performed as previously described [21].The primer pairs Cgrcon7F-Cgrcon7R and Cscon7F-Cscon7R were used for amplifying the open reading frames (ORFs) of Cgrcon7 and Cscon7 from the cDNAs, respectively.The protein domain was analyzed on the SMART platform (http://smart.embl-heidelberg.de/,accessed on 18 March 2024).The BLAST tool (https: //blast.ncbi.nlm.nih.gov/Blast.cgi,accessed on 18 March 2024) in GenBank was used to search Con7 homologous protein sequences in different fungi.Protein sequence alignment and motif analysis were carried out as previously described [22].

Target Disruption and Complementation
Cgrcon7 and Cscon7 were disrupted using the homologous recombination method (Figure S1).For deleting Cgrcon7, the primer pairs Cgrcon7UF-Cgrcon7UR and Cgrcon7DF-Cgrcon7DR were used to amplify 1108 bp-upstream and 1003 bp-downstream flanking fragments from the gDNA of C. graminicola, respectively.The two fragments were ligated to a vector pUC18-HPT (pUC18 harboring the hygromycin phosphotransferase gene HPT).Then, the linearized plasmid was transformed into CgrWT protoplasts and the transformation was performed as described previously [23].Transformants were screened and verified by PCR using the primer pairs Cgrcon7F-Cgrcon7R, Cgrcon7UU-pUC18-PI and pUC18-PI1-Cgrcon7DD (Figure S1A).The primers Cgrcon7hbF and Cgrcon7hbR were used to amplify the complementary fragment of Cgrcon7, which was inserted into the pUC18 vector fused with a neomycin resistance gene.The complementary vector was transformed into protoplasts derived from a Cgrcon7-deletion mutant.Then, transformants were selected on a medium containing G418, and the primers Cgrcon7F and Cgrcon7R were used for PCR validation.
The gene deletion and complementation of Cscon7 were performed as previously described [21].Briefly, the 5 ′ and 3 ′ flank sequences of Cscon7 were inserted into pCB1532 and then transformed into protoplasts from C. siamense CsWT.Transformants were screened and validated using Cscon7F-Cscon7R, Cscon7UU-PI, and PI1-Cscon7DD (Figure S1B).The complementary fragment of Cscon7 was inserted into the pUC18-HPT vector and then transformed into protoplasts prepared from a Cscon7-deletion mutant.Then, transformants were screened on the TB3 agar medium containing hygromycin B, and Cscon7F and Cscon7R were used for PCR verification.The primers used in this section are listed in Table S1.

Subcellular Localization
To analyze the subcellular localization of CgrCon7, the trpC promoter, Cgrcon7-coding sequence, eGFP-coding sequence, and trpC terminator were fused and inserted into the vector pUC18-HPT.The recombinant plasmid was transformed into protoplasts from strain CgrWT.For C. siamense, the construction of the Cscon7-eGFP fusion fragment was similar to that of Cgrcon7.The fragment was ligated to pCB1532 and transformed into protoplasts from strain CsWT.The transformation was performed exactly as described previously [24].The mycelia and conidia from transformants were observed using a fluorescent microscope (Leica DM LB2, Leica Microsystems Inc., Wetzlar, Germany).Nuclei were visualized using 4 ′ ,6-diamidino-2-phenylindole (DAPI) (10 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA).

Stress Response Assays
To test the cell wall integrity (CWI), mycelial plugs from the wild type, gene-deletion mutants, and complementary strains were inoculated on MM plates with SDS and congo red (CR).The colony diameter was determined on 7 dpi, and inhibition rates were calculated as described previously [19].For calcium fluorescent white (CFW) staining, the mycelial plugs from different strains were placed on glass slides and incubated at 28 • C.After 3 days, hyphae were stained with CFW staining solution containing 1 g/L CFW and 1 M NaOH (v/v = 1:1) for 5 min in the dark and visualized using fluorescence microscopy [22].
For H 2 O 2 -sensitive assays, strains were incubated in the PDB medium at 28 • C and conidia were obtained by filtration on 3 dpi.Fifteen milliliters of PDA medium was mixed with 100 µL conidial suspension (1 × 10 6 conidia/mL) to pour a plate.Then, a filter paper disk soaked with 30% H 2 O 2 was placed in the center of the plate.The plated was cultured at 28 • C, and the diameter of the inhibition zone was determined [16].

Conidiation, Germination, and Appressorium Formation
To obtain falcate conidia of C. graminicola, the strains were cultured on oatmeal agar (OMA: 50 g/L oatmeal, 18 g/L agar) for 14 days at 28 • C. The falcate conidia were rinsed using 5 mL of sterile water, and the conidial yield was determined using a hemocytometer.Regarding oval conidia, the strains were cultured in PDB for 48 h at 28 • C. Miracloth (Calbiochem, San Diego, CA, USA) was used for removing hypha of the sampled culture, and conidial yields were counted using the hemocytometer.The collection and determination of conidia from C. siamense were similar to those of oval conidia from C. graminicola.
For conidial germination, 20 µL of conidial suspension (5 × 10 5 conidia/mL) was dropped on cellophane and kept at 28 • C.Then, conidial germination and appressorium formation were observed under a microscope.The percentages of germination or appressorium formation were determined at desired time points.To observe the formation of hyphopodia, mycelial plugs were inoculated onto glass slides and cultured at 28 • C, and the hyphopodium was observed using a microscope on 3 dpi.

Virulence Assay
The inoculation assay of C. graminicola strains was performed using susceptible maize (Zea mays L.) cultivar Xianyu 335.Three-week-old maize leaves were selected and wounded using a sterile needle.Mycelial plugs (5 mm diameter) or 20 µL of falcate conidial suspensions (5 × 10 5 conidia/mL) of the strains were inoculated onto maize leaves, and mock inoculation was performed with agar plugs or sterile water, respectively.The virulence assay of C. siamense was similar to that of C. graminicola, except for using detached rubber tree leaves as inoculation objects.The leaves were kept in a moist chamber (relative humidity > 90%), and the severity of disease symptoms was recorded on 3 dpi.

RNA Sequencing
Transcriptome analysis of ∆Cgrcon7 (∆Cscon7) and CgrWT (CsWT) was performed by RNA sequencing (RNA-Seq).For C. graminicola, the wild type and ∆Cgrcon7 were cultured on PDA medium at 28 • C for 5 days, and their mycelia were collected to extract total RNAs.Both the CgrWT and ∆Cgrcon7 samples had three repeats.The RNA-Seq was performed by BGI (Wuhan, China) using a MGISEQ-2000 platform.The genome of C. graminicola M1.001 from GenBank was used as a reference.Data processing and analysis were based on a previous method [21].The RNA-Seq of Cscon7 was similar to that of Cgrcon7, and the genome of C. siamense Cg363 from GenBank was used as a reference.The RNA-Seq data were verified using quantitative RT-PCR (qRT-PCR) [25].

Statistical Analysis
All experiments were performed in triplicate, and the results are expressed as the mean ± standard deviation.SPSS software 20.0 was used for data analyses.One-way ANOVA test and Duncan's multiple comparisons were used for significant difference analysis (p < 0.05 and p < 0.01), and GraphPad Prism 8.0 was used for graph making.
Fluorescence microscopy examination displayed that both CgrCon7 and CsCon7 were mainly localized in the nucleus of hyphae, conidia, and appressoria, with a small distribution in the cytoplasm (Figure 2A,B).The expression levels of Cgrcon7 and Cscon7 were investigated by qRT-PCR at different growth stages.With the increase in culture time, the expression levels of two genes increased continuously.Furthermore, both Cgrcon7 and Cscon7 exhibited high expression levels in conidia (Figure S2A,B).Fluorescence microscopy examination displayed that both CgrCon7 and CsCon7 were mainly localized in the nucleus of hyphae, conidia, and appressoria, with a small distribution in the cytoplasm (Figure 2A,B).The expression levels of Cgrcon7 and Cscon7 were investigated by qRT-PCR at different growth stages.With the increase in culture time, the expression levels of two genes increased continuously.Furthermore, both Cgrcon7 and Cscon7 exhibited high expression levels in conidia (Figure S2A,B).

Gene Disruption and Complementation of Cgrcon7 and Cscon7
To study the biological function of CgrCon7 and CsCon7, we generated Cgrcon7and Cscon7-knockout mutants using the homologous replacement method.For the deletion of Cgrcon7 in C. graminicola, the validation results displayed that transformants 10, 13, and 17 had a specific amplification band using Cgrcon7UU-pUC18-PI and pUC18-PI1-Cgrcon7DD (Figure S3B,C), whereas no target band was amplified using Cgrcon7F-

Gene Disruption and Complementation of Cgrcon7 and Cscon7
To study the biological function of CgrCon7 and CsCon7, we generated Cgrcon7and Cscon7-knockout mutants using the homologous replacement method.For the deletion of Cgrcon7 in C. graminicola, the validation results displayed that transformants 10, 13, and 17 had a specific amplification band using Cgrcon7UU-pUC18-PI and pUC18-PI1-Cgrcon7DD (Figure S3B,C), whereas no target band was amplified using Cgrcon7F-Cgrcon7R (Figure S3A).The three transformants were named ∆Cgrcon7-10, ∆Cgrcon7-13 and ∆Cgrcon7-17, respectively.Regarding the disruption of Cscon7 in C. siamense, two Cscon7-knockout mutants were obtained using the same method and named ∆Cscon7-4 and ∆Cscon7-11 (Figure S3D-F).For complementation, the gDNA sequences of Cgrcon7 and Cscon7 were retransformed into their corresponding disruption mutants, and their complementary strains were named ∆Cgrcon7-C and ∆Cscon7-C, respectively.

Con7 Is Required for Vegetative Growth in C. graminicola and C. siamense
The strains were cultured on PDA, CM, CZ, and MM media to evaluate vegetative growth.As shown in Figure 3, all deletion mutants of Cgrcon7 and Cscon7 decreased in growth rate significantly on the four media.For instance, the diameter of ∆Cgrcon7 was about 2.6 cm on the CZ medium, whereas the diameter of CgrWT was up to 6.6 cm.The growth rates of ∆Cgrcon7 and ∆Cscon7 were less than 50% those of the wild-type strains on CZ and MM media.These results show that Con7 is involved in regulating the vegetative growth of C. graminicola and C. siamense.To investigate the role of CgrCon7 in CWI, all the strains were cultured on media supplemented with SDS and CR.The results showed that the Cgrcon7-deletion mutants

Con7 Regulates CWI and Oxidative Stress in C. graminicola and C. siamense
To investigate the role of CgrCon7 in CWI, all the strains were cultured on media supplemented with SDS and CR.The results showed that the Cgrcon7-deletion mutants were more sensitive to SDS and CR than CgrWT (Figure 4A,B).∆Cscon7 was similar to ∆Cgrcon7, exhibiting elevated sensitivity to two stress factors (Figure 4C,D).Furthermore, the distribution of chitin was investigated by CFW dyeing in C. graminicola.In the hyphae and conidia of CgrWT, chitin was located uniformly in the cell wall and concentrated in the septa of hyphae.In contrast, chitin displayed enhanced distribution in the cell wall of ∆Cgrcon7 and also exhibited punctate distribution in some areas of hyphae and falcate conidia (Figure 4E).The above results suggest that CgrCon7 and CsCon7 participate in the regulation of cell wall integrity.
The H 2 O 2 -sensitivity assay was used to test whether Con7 affects oxidative stress in C. graminicola and C. siamense.As shown in Figure 5A,B, the filter paper had formed apparent inhibition zones on all the plates inoculated with different strains.The diameters of inhibition zones caused by ∆Cgrcon7 and ∆Cscon7 were significantly larger than those of the wild-type strains (Figure 5C,D).Deleting Cgrcon7 or Cscon7 resulted in apparent defects in H 2 O 2 detoxification.Therefore, Con7 is involved in the regulation of the oxidative stress response in C. graminicola and C. siamense.

Con7 Is Involved in Conidiation, Conidial Morphology, Appressorium, and Hyphopodium Formation
For falcate conidia, the C. graminicola strains were cultured on OMA medium, a the conidial production was determined on 14 dpi.It was found that the yield of falc conidia from ΔCgrcon7 was about 1% that of CgrWT (Figure 6A).We further determin the oval conidium yield of ΔCgrcon7, which was also significantly lower than that CgrWT (Figure 6B).Moreover, deleting Cgrcon7 made the falcate conidium more slend and the falcate conidium of ΔCgrcon7 became longer and thinner than that of CgrW (Figure S4A,B).There was no dramatic difference in the germination of falcate conidia oval conidia between ΔCgrcon7 and CgrWT.However, the falcate conidia from ΔCgrc did not form appressoria normally on cellophane, whereas CgrWT had produ melanized appressoria at 12 h postinoculation (Figure 6C).

Con7 Is Involved in Conidiation, Conidial Morphology, Appressorium, and Hyphopodium Formation
For falcate conidia, the C. graminicola strains were cultured on OMA medium, and the conidial production was determined on 14 dpi.It was found that the yield of falcate conidia from ∆Cgrcon7 was about 1% that of CgrWT (Figure 6A).We further determined the oval conidium yield of ∆Cgrcon7, which was also significantly lower than that of CgrWT (Figure 6B).Moreover, deleting Cgrcon7 made the falcate conidium more slender, and the falcate conidium of ∆Cgrcon7 became longer and thinner than that of CgrWT (Figure S4A,B).There was no dramatic difference in the germination of falcate conidia or oval conidia between ∆Cgrcon7 and CgrWT.However, the falcate conidia from ∆Cgrcon7 did not form appressoria normally on cellophane, whereas CgrWT had produced melanized appressoria at 12 h postinoculation (Figure 6C).
As for C. siamense, deleting Cscon7 also led to distinctly decreased conidial production, and ∆Cscon7 exhibited lower germination rates than CsWT (Figure 7A,B).Furthermore, ∆Cscon7 did not produce appressoria, the same as ∆Cgrcon7 (Figure 7C,D).The conidia of ∆Cscon7 were smaller in length and width than those of CsWT (Figure S4C,D).We further detected the formation of hyphopodia in C. graminicola and C. siamense, and the results showed that the mycelia of ∆Cgrcon7 and ∆Cscon7 did not form hyphopodia normally on glass slides (Figure S5A,B).These findings suggest that Con7 is required for conidiation, maintaining conidial morphology, appressorium, and hyphopodium formation in C. graminicola and C. siamense.As for C. siamense, deleting Cscon7 also led to distinctly decreased conidial production, and ΔCscon7 exhibited lower germination rates than CsWT (Figure 7A,B).Furthermore, ΔCscon7 did not produce appressoria, the same as ΔCgrcon7 (Figure 7C,D).The conidia of ΔCscon7 were smaller in length and width than those of CsWT (Figure S4C,D).We further detected the formation of hyphopodia in C. graminicola and C. siamense, and the results showed that the mycelia of ΔCgrcon7 and ΔCscon7 did not form hyphopodia normally on glass slides (Figure S5A,B).These findings suggest that Con7 is required for conidiation, maintaining conidial morphology, appressorium, and hyphopodium formation in C. graminicola and C. siamense.

Con7 Is Required for the Virulence of C. graminicola and C. siamense
To determine the role of Cgrcon7 in the virulence of C. graminicola, the CgrWT, ΔCgrcon7, and complementary strains were inoculated on maize leaves.Regardless of the inoculation mode, typical and enlarged lesions were observed on the maize leaves inoculated with the wild type and complementary strain on 3 dpi.In contrast, there were limited lesions on the leaves inoculated with the ΔCgrcon7 mutants, and the lesion size showed no significant difference from that of the control group (Figure 8A-C).Subsequently, we determined the virulence of ΔCscon7 on rubber tree leaves.Compared with

Con7 Is Required for the Virulence of C. graminicola and C. siamense
To determine the role of Cgrcon7 in the virulence of C. graminicola, the CgrWT, ∆Cgrcon7, and complementary strains were inoculated on maize leaves.Regardless of the inoculation mode, typical and enlarged lesions were observed on the maize leaves inoculated with the wild type and complementary strain on 3 dpi.In contrast, there were limited lesions on the leaves inoculated with the ∆Cgrcon7 mutants, and the lesion size showed no significant difference from that of the control group (Figure 8A-C).Subsequently, we determined the virulence of ∆Cscon7 on rubber tree leaves.Compared with CsWT, ∆Cscon7 did not form visible lesions on leaves and lost virulence completely (Figure 8D-F).These results indicate that Con7 is essential for the virulence of C. graminicola and C. siamense.

Transcriptomic Analysis of CgrCon7 and CsCon7
In order to analyze the regulatory network of CgrCon7 (CsCon7), the global RNA expression profiles of ΔCgrcon7 (ΔCscon7) and CgrWT (CsWT) were compared.The selection for differentially expressed genes (DEGs) was established as a minimum of 2.0-fold down-or upregulation in the deletion mutant versus the wild type.For CgrCon7, 2907 genes were identified as DEGs, including 1588 upregulated genes and 1319 downregulated genes.Regarding CsCon7, 4124 genes displayed significant differences in expression levels, in which 1220 genes were upregulated and 2904 genes were downregulated.The RNA-Seq results of ΔCgrcon7 and ΔCscon7 were validated by qRT-PCR, and the expression levels of selected DEGs exhibited the same trend as those in RNA-Seq data, with all the correlation coefficients being greater than 96% (Figure S6).
Then, we performed GO (Gene Ontology) function and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses of DEGs from ΔCgrcon7 and ΔCscon7.It was found that the functions of DEGs from two deletion mutants were mainly classified in (over 200 DEGs) transport and catabolism, signal transduction, translation, amino acid metabolism, carbohydrate metabolism, global and overview maps, and lipid metabolism (Figure 9A,C).The top 20 significantly enriched KEGG terms are listed in Figure 9B,D.

Transcriptomic Analysis of CgrCon7 and CsCon7
In order to analyze the regulatory network of CgrCon7 (CsCon7), the global RNA expression profiles of ∆Cgrcon7 (∆Cscon7) and CgrWT (CsWT) were compared.The selection for differentially expressed genes (DEGs) was established as a minimum of 2.0-fold downor upregulation in the deletion mutant versus the wild type.For CgrCon7, 2907 genes were identified as DEGs, including 1588 upregulated genes and 1319 downregulated genes.Regarding CsCon7, 4124 genes displayed significant differences in expression levels, in which 1220 genes were upregulated and 2904 genes were downregulated.The RNA-Seq results of ∆Cgrcon7 and ∆Cscon7 were validated by qRT-PCR, and the expression levels of selected DEGs exhibited the same trend as those in RNA-Seq data, with all the correlation coefficients being greater than 96% (Figure S6).Then, we performed GO (Gene Ontology) function and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses of DEGs from ∆Cgrcon7 and ∆Cscon7.It was found that the functions of DEGs from two deletion mutants were mainly classified in (over 200 DEGs) transport and catabolism, signal transduction, translation, amino acid metabolism, carbohydrate metabolism, global and overview maps, and lipid metabolism (Figure 9A,C).The top 20 significantly enriched KEGG terms are listed in Figure 9B,D.The DEGs from ∆Cgrcon7 and ∆Cscon7 were both enriched in ribosome, fatty acid degradation, fatty acid metabolism, starch and sucrose metabolism, galactose metabolism, beta-alanine metabolism, valine, leucine, and isoleucine degradation, and glyoxylate and dicarboxylate metabolism.It was also noted that some enzyme genes related to ROS (reactive oxygen species) detoxification, chitin synthesis, and cell wall degradation were significantly affected due to the deletion of Cgrcon7 or Cscon7, including superoxide dismutase, peroxidase, catalase, chitin synthase, cutinase, pectin lyase, glucanase, etc. (Table 1).

Discussion
The C 2 H 2 -type zinc finger is the most common DNA-binding motif in eukaryotes, especially in C 2 H 2 TFs [28].C 2 H 2 TFs play an important role in regulating different signal transduction pathways and controlling various biological processes in eukaryotic cells.In this study, we identified and characterized two C 2 H 2 zinc finger proteins, CgrCon7 and CsCon7, in C. graminicola and C. siamense that were orthologous to Con7p in M. oryzae.Con7-like TFs widely exist in filamentous fungi, but not in yeasts, suggesting that they may coordinate certain functions common to molds [15].The spatiotemporal expression analysis showed that CgrCon7 and CsCon7 were mainly localized in the nucleus and expressed throughout the whole vegetative growth process, with high expression levels in conidia.The expression patterns suggest that CgrCon7 and CsCon7 may play crucial roles in the development of hyphae and conidia.
To investigate the functions of CgrCon7 and CsCon7, we constructed their deletion mutants in C. graminicola and C. siamense, respectively.Phenotypic analysis revealed that ∆Cgrcon7 and ∆cscon7 both exhibited notably decreased vegetative growth compared with the wild-type strains.The Con7 orthologs also displayed a positive regulatory role in the growth of Verticillium dahliae, F. oxysporum, and F. graminearum [16,17,29].Based on the RNA-Seq results, we found that a large number of genes associated with the metabolism of carbohydrates, lipids, and amino acids were significantly influenced in ∆Cgrcon7 and ∆Cscon7.Therefore, deleting Cgrcon7 or Cscon7 may cause metabolic disorders and further affect the vegetative growth of two Colletotrichum species.In V. dahliae, the RNA-Seq data showed that 34.5% of the downregulated genes in the VTA2-deletion mutant were involved in metabolism-related processes [16].Moreover, numerous DEGs in the ∆con7-1 mutant were also found to participate in regulating carbohydrate metabolism (133 genes), fatty acid metabolism (49), and amino acid metabolism (64) [17].
The fungal cell wall, which is typically composed of chitin and glucan, plays a crucial role in growth and development as well as adaptation to adverse environments [30,31].In our study, deleting Cgrcon7 (Cscon7) resulted in elevated sensitivity against SDS and CR, suggesting that Con7 is involved in maintaining CWI in C. graminicola and C. siamense.The KEGG analysis showed that the expression of over 300 genes in the mitogen-activated protein kinase (MAPK) signaling pathway were dramatically affected in ∆Cgrcon7 or ∆Cscon7, and many of them were related to the CWI pathway.Furthermore, the CFW dyeing results showed that there were obvious differences in chitin distribution between ∆Cgrcon7 and CgrWT, suggesting CgrCon7 may be involved in regulating chitin synthesis.The RNA-Seq data showed that the expression of two chitin synthase genes (GLRG_05787 and GLRG_03399) was affected to varying degrees by CgrCon7, and three chitin synthase genes were upregulated in the Cscon7-deletion mutant.Con7-like TFs have been reported to be connected with chitin metabolism.In M. oryzae, Con7p was involved in regulating the expression of genes related to cell wall biogenesis and remodeling, including the class VI chitin synthase Chs7 and the chitin-binding proteins Cbp1 and Cbp2 [32].Furthermore, the chitin synthase gene (FGSG_06550) was significantly upregulated in the FgCON7-deletion mutant and affected the cell wall integrity of F. graminearum [18].Overall, we speculate that Con7 participates in the regulation of chitin synthesis and further affects CWI in C. graminicola and C. siamense.
Conidia are important tools for the dissemination and infection of C. graminicola and C. siamense, and Con7 plays a crucial role in conidial production in the two pathogenic fungi.Disrupting Cgrcon7 led to dramatically reduced conidiation, and the yield of falcate conidia of ∆Cgrcon7 was only 1% of that of the wild type.Similar phenotypes also occurred in the Cscon7-deletion mutant.It has been reported that the Con7-like TF acts as a typical positive regulator in asexual sporulation in M. oryzae and Verticillium and Fusarium species [16,18,29,33].From the RNA-Seq result of CgrCon7, we found that two regulatory genes of asexual development, CgrabaA (GLRG_00681) and CgrwetA (GLRG_04344), were downregulated in ∆Cgrcon7.Moreover, it was also observed that CsbrlA (CGCS363_v003651) was significantly downregulated in ∆Cscon7.In Aspergillus nidulans, three regulators, BrlA, AbaA, and WetA, constitute a central regulatory pathway of conidiogenesis, which can sequentially activate conidial production and mediate the expression of specific genes related to asexual development [34].Therefore, we speculate that Con7 may affect conidiation by regulating the conidial developmental pathway BrlA-AbaA-WetA in C. graminicola and C. siamense.In F. graminearum, it has been proven that there is a direct genetic link between FgCON7 and FgABAA, and the expressions of FgABAA and FgWETA were significantly decreased in ∆Fgcon7 [18].
In addition to the decrease in conidiation, ∆Cgrcon7 and ∆Cscon7 also exhibited changes in conidial morphology.We speculate that deleting con7 may influence chitin biosynthesis and alter the CWI of conidia, which further affects the shape of conidia in C. graminicola and C. siamense.Another remarkable phenotype is that disrupting Cgr-con7 and Cscon7 completely abolishes the ability of appressorium formation as well as hyphopodium development.In M. oryzae, Con7p is also required for appressorium formation, and the con7-deletion mutant failed to develop appressoria, probably due to the defect of chitin accumulation [15].From the RNA-Seq result of ∆Cgrcon7, we noticed that a homeobox transcription factor (GLRG_00169), an ortholog of MoHox7 (Pth12) in M. oryzae, was downregulated in ∆Cgrcon7 [35].The mutant ∆Mohox7 could not produce appressoria (hyphopodia) at the tips of germ tubes and hyphae, leading to a loss of pathogenicity [35].CgrCon7 may be involved in regulating the expression of GLRG_00169 and further driving the appressorium formation of C. graminicola.
In the pathogenicity assay, both ∆Cgrcon7 and ∆Cscon7 exhibited complete loss of virulence on maize and rubber tree leaves, respectively.Con7p and Con7-1 also displayed comparable roles in the pathogenicity of M. oryzae and F. oxysporum [15,17].Given the critical role of appressoria in penetration, the defect in appressorium formation may be the main cause of the non-pathogenicity of ∆Cgrcon7 and ∆Cscon7.In addition, deleting con7 led to increased sensitivity to H 2 O 2 , suggesting that Con7 may be involved in H 2 O 2 detoxification.The transcriptome analysis showed that some genes related to ROS detoxification, such as superoxide dismutase, peroxidase, and catalase, were significantly downregulated in ∆Cgrcon7 and ∆Cscon7 (Table 1).In C. siamense, the transcription factor gene CgAP1 is also downregulated by CgCon7, which has been reported to play a vital role in the oxidative stress and pathogenicity of C. gloeosporioides (Table 1) [21].Therefore, Con7 can also affect the pathogenicity through coordinating ROS detoxification in C. graminicola and C. siamense.From the RNA-Seq results, we also found that the expressions of several enzyme genes related to cell wall degradation were notably downregulated in ∆Cgrcon7 and ∆Cscon7 (Table 1).Plant pathogenic fungi usually secrete various hydrolases, such as cutinase, pectinase, and cellulose, to degrade the cell wall of hosts, contributing to their infection.Overall, our results suggest that Con7 may influence the pathogenicity of C. graminicola and C. siamense by regulating asexual sporulation, appressorium formation, ROS detoxification, and the expression of cell wall-degrading enzymes, etc.
In summary, Con7 (CgrCon7 and CsCon7) acts as a global transcription factor in C. graminicola and C. siamense, and is involved in regulating vegetative growth, CWI, oxidative stress, asexual development, appressorium and hyphopodium formation, and virulence.Future work will focus on the exploration of genes directly regulated by CgrCon7 or CsCon7, further revealing their regulatory networks and mechanisms.

17 Figure 3 .
Figure 3. Vegetative growth on four media.(A) Growth comparison of C. graminicola strains on four media.(B) Statistical analyses of colony diameters of C. graminicola strains.(C) Growth comparison of C. siamense strains on four media.(D) Statistical analyses of colony diameters of C. siamense strains.** Significant at p < 0.01.

Figure 3 .
Figure 3. Vegetative growth on four media.(A) Growth comparison of C. graminicola strains on four media.(B) Statistical analyses of colony diameters of C. graminicola strains.(C) Growth comparison of C. siamense strains on four media.(D) Statistical analyses of colony diameters of C. siamense strains.** Significant at p < 0.01.

Figure 4 .
Figure 4. Effects of Con7 on the cell wall integrity of C. graminicola and C. siamense.(A) Effects of SDS and congo red (CR) on the growth of C. graminicola strains.(B) Statistical analyses of inhibition rates of C. graminicola strains.(C) Effects of SDS and CR on the growth of C. siamense strains.(D) Statistical analyses of inhibition rates of C. siamense strains.* Significant at p < 0.05; ** significant at p < 0.01.(E) CFW dyeing of the mycelia and falcate conidia of C. graminicola strains.s: septa; ch: chitin.The H2O2-sensitivity assay was used to test whether Con7 affects oxidative stress in C. graminicola and C. siamense.As shown in Figure 5A,B, the filter paper had formed apparent inhibition zones on all the plates inoculated with different strains.The diameters of inhibition zones caused by ΔCgrcon7 and ΔCscon7 were significantly larger than those

Figure 4 .
Figure 4. Effects of Con7 on the cell wall integrity of C. graminicola and C. siamense.(A) Effects of SDS and congo red (CR) on the growth of C. graminicola strains.(B) Statistical analyses of inhibition rates of C. graminicola strains.(C) Effects of SDS and CR on the growth of C. siamense strains.(D) Statistical analyses of inhibition rates of C. siamense strains.* Significant at p < 0.05; ** significant at p < 0.01.(E) CFW dyeing of the mycelia and falcate conidia of C. graminicola strains.s: septa; ch: chitin.

Figure 5 .
Figure 5. H2O2-sensitive assays.(A) Effects of H2O2 on the growth of C. graminicola strains on 3 (B) Effects of H2O2 on the growth of C. siamense strains on 2 dpi.(C) Statistical analyses of inhibi zone diameters of C. graminicola strains.(D) Statistical analyses of inhibition zone diameters o siamense strains.** Significant at p < 0.01.

Figure 5 .
Figure 5. H 2 O 2 -sensitive assays.(A) Effects of H 2 O 2 on the growth of C. graminicola strains on 3 dpi.(B) Effects of H 2 O 2 on the growth of C. siamense strains on 2 dpi.(C) Statistical analyses of inhibition zone diameters of C. graminicola strains.(D) Statistical analyses of inhibition zone diameters of C. siamense strains.** Significant at p < 0.01.

Figure 9 .
Figure 9. Functional classification and KEGG enrichment analyses of DEGs.(A) Functional classification of DEGs in the Cgrcon7-deletion mutant.(B) The top 20 enriched KEGG pathways of DEGs in the Cgrcon7-deletion mutant.(C) Functional classification of DEGs in the Cscon7-deletion mutant.(D) The top 20 enriched KEGG pathways of DEGs in the Cscon7-deletion mutant.

Figure 9 .
Figure 9. Functional classification and KEGG enrichment analyses of DEGs.(A) Functional classification of DEGs in the Cgrcon7-deletion mutant.(B) The top 20 enriched KEGG pathways of DEGs in the Cgrcon7-deletion mutant.(C) Functional classification of DEGs in the Cscon7-deletion mutant.(D) The top 20 enriched KEGG pathways of DEGs in the Cscon7-deletion mutant.

Table 1 .
Expression of some DEGs affected by CgrCon7 and CsCon7.

Table 1 .
Expression of some DEGs affected by CgrCon7 and CsCon7.